Introduction
Herpesvirus-like sequences, designated KS330 [1], have been detected in AIDS-associated Kaposi's sarcoma (KS) lesions, and have been found to belong to a new human herpesvirus named HHV-8 or KS-associated herpesvirus (KSHV) [2]. Their occurrence in lesions of classic and endemic KS [3,4], both unrelated to HIV infection, together with previous epidemiological observations [5,6] are suggestive of an aetiological role of this novel agent in all of these forms of neoplasia. These observations do not permit the elaboration of a unified theory of the pathogenesis that accounts for the many features of KS and do not clarify how this herpesvirus spreads in different human populations.
The most important distinguishing characteristic between the classical presentation of KS and AIDS-related KS is the more aggressive biological behaviour typically seen in young immunosuppressed patients. Thus further factors, in addition to immunosupression which probably accelerates this complex disorder, must be taken into consideration. It is relevant to discriminate between our knowledge of the normal dissemination of HHV-8 in epidemiological settings such as some Mediterranean and African areas, where KS was present before the epidemiological spread of HIV, and our knowledge of other countries where the HIV and AIDS-related KS appeared 15 years ago.
According to reported data, incidence rates of KS in Italy [7], before AIDS spread, were 1.0 and 0.3 per 100 000 in male and female groups, respectively. Significantly higher values (threefold higher in males and twofold higher in females) were recorded in Sicily, where spread of this neoplasia in the pre-AIDS era was 10-fold greater than in the US and more than 50-fold greater than in England and Wales [7]. On the assumption that studies carried out in a high-endemic KS area, such as Sicily, can contribute to elucidating the natural history of HHV-8 infection, we investigated the presence of HHV-8 DNA sequences in skin biopsies, peripheral blood mononuclear cells (PBMC), sperm and cervical swabs taken from heterosexual HIV-positive and HIV-negative individuals affected or unaffected by KS.
Materials and methods
Study samples and tissue preparation
A retrospective study was carried out on paraffin-embedded biopsies of skin of 24 classic KS, four AIDS-associated KS, and 12 skin samples, taken from different pathological specimens (five melanocytic nevus, one angiokeratoma, two angioma, one granuloma pyogenicum, one lentigo maligna and two seborrheic keratosis) and five normal skin specimens, as controls. Sections 10 µm thick, from paraffin-embedded samples, were treated and DNA was extracted (from one to three sections for each sample) according to described procedures [8]. As a control for the adequacy of genomic DNA extraction, from 5 to 10 µl of the extracted samples were analysed using the polymerase chain reaction (PCR) for human β-globin gene. Of all samples extracted, two classic KS and two pathological skin samples (one angioma and one angiokeratoma) were classified as β-globin negative and therefore excluded from the study.
PBMC were obtained from four individuals with classic KS, four with AIDS-associated KS and 30 AIDS patients without KS; a semen sample was also collected from one patient with classical KS and 10 AIDS patients. Both sperm and PBMC samples were also obtained from 25 healthy HIV-negative donors (age range, 22-40 years) who were enrolled from among students, healthcare workers and from 20 male HIV-negative subjects (age range, 27-50 years), who had, or had a previous history of, sexually transmitted diseases (STD). Finally, samples of exfoliated cervical cells, obtained from the uterine ectocervices of 47 sexually active HIV-negative women (age range, 19-50 years), were included in the study.
DNA extracted from PBMC [9], sperm [10] and cervical swabs [11] were positive for PCR amplification of a β-globin DNA fragment.
PCR
A two-step amplification method for the detection of HHV-8 DNA was performed according to a previously described technique [12]. Two sets of commercially available primers were used, KS1 and KS2, described by Chang et al. [1], which target a sequence of 233 bp DNA fragment (KS330 Bam) from a capsid protein gene and NS1 and NS2, which amplify a 160 bp fragment within the region of HHV-8 genome mentioned above. The first step PCR was done with a 1 µg of sample DNA (PBMC and semen), or a volume of the lysate samples (skin and cervical swabs) which was positive for β-globin assay.
After the first-step amplification, 10 µl of the PCR product was further amplified with the inner primers. Amplified products were analysed by 12% acrylamide-bis-acrylamide gel electrophoresis. To avoid possible contamination of PCR mixtures, all reactions were done under stringent conditions [13]. For each run, blank mixtures with no or unrelated DNA were interspersed with the samples. The positive control consisted of 233 bp product obtained after first-step PCR amplification of a KS bioptic sample. The positive band was purified with an extraction kit (Qiaquick-spin PCR purification kit; Qiagen Ag, Basel, Switzerland) and its concentration estimated by spectrophotometric readings. The sensitivity of the two-step reaction was determined by serial dilution of a known amount of the 233 bp purified product. About five target molecules were detected after ethidium bromide staining, according to the data reported by Bigoni et al. [14] and P. Monini (personal communication, 1996).
Furthermore, in order to exclude the possibility that the positive results of the semen samples were due to laboratory contamination, these specimens were processed for DNA extraction and tested in separate laboratories by a second set of non-overlapping primers, which amplified a region of a KS631 Bam sequences. Thus, the first step was performed by using a set of external primers (081-082), which amplified 600 bp, as previously described by Gessain et al. [15], whereas the second inner primers (0891-0892) corresponded to a fragment of 253 bp (M. Corbellino, personal communication, 1996).
Each PCR reaction was performed in a final volume of 100 µl, containing 200 µM deoxyribonucleoside triphosphate, a 0.2 µM concentration of each HHV-8 specific primer, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris, and 2.5 units AmpliTaq DNA polymerase (Perkin Elmer Cetus, Norwalk, Connecticut, USA). Both PCR steps were performed with an initial denaturation step at 94°C for 2 min, followed by 10 cycles at 94°C for 30 sec and 60°C for 30 sec. The next 35 cycles consisted of 10 sec incubation time at 94°C, and 60°C for 30 sec.
Results
Sarcoma tissue was available from 22 patients with classic KS and four with AIDS-associated KS. The first group was composed of 11 men and 11 women with a median age of 69 years (range 42-82 years); none of them reported a history of intravenous drug use and all were HIV-seronegative at the time of biopsy. No males reported homosexual intercourse. The four patients with AIDS-associated KS were all intravenous drug users. Their median age at the time of the diagnosis was 29 years (age range, 28-32 years) and they had a depressed CD4 T-cell counts (median count 95 × 106/l, range 16-140 cells × 106/l), whereas patients with classic KS had normal counts.
Twenty out of 22 samples from classic KS and all tissue samples from AIDS-associated KS were positive by two-step PCR and gave a signal of the expected size. Seventeen from the former group and three out of four of the latter were also positive by one-step PCR performed with the primers KS1 and KS2 [1], however. None of the control specimens was positive by two-step PCR (Table 1). The identity of the amplified DNA sequences was verified by digestion with restriction enzymes PstI and NcoI, cutting once within the viral sequences amplified [12].
We further investigated the presence of HHV-8 in PBMC of individuals with classic KS, HIV-infected individuals with and without KS and healthy normal donor controls. The presence of HHV-8 DNA was found in two out of four (50%) individuals with classic KS and in two out of four patients with AIDS-associated KS (50%) and in three (10%) of 30 persons with AIDS without KS. A similar prevalence value of 11% was also observed in samples taken from 45 healthy PBMC donors (Table 2).
All the PBMC from patients with KS and AIDS-associated KS gave positive results when submitted to one-step PCR only. Similar results (two out of three positive) were obtained with the PBMC of AIDS patients without KS. On the contrary, the positive samples from healthy donors were negative to one-step PCR and gave faint bands only after a two-step PCR.
During this study we also collected a semen sample from one subject with classic KS, 10 AIDS patients and 45 healthy individuals whose PBMC had been analysed. Because detection of HHV-8 DNA in semen of HIV-negative individuals is controversial so far [12,16-19], these samples were also tested with a second nested PCR, which utilizes a set of non-overlapping primers of different HHV-8 DNA sequences (KS631 Bam).
HHV-8 DNA sequences were detected in semen sample from the patient with classic KS, from one out of 10 AIDS patients (10%) and from six out 45 (13%) healthy donors with and without STD. The positive results were detected only after the second step of both PCR methods (Table 3). It should be stressed that the patient with classical KS, the AIDS patient without KS and the five healthy HIV-negative individuals carried HHV-8 DNA in both semen and PBMC samples. To ascertain whether the presence of HHV-8 was directly correlated with heterosexual activity, cervical swabs taken from women with normal and abnormal cytology were also tested for the presence of HHV-8 DNA. As Table 3 shows, no HHV-8 DNA fragments were detected in cervical swabs.
Discussion
Data recorded by the Sicilian cancer register indicates that KS is present in the island with a high incidence rate in both sexes [7], and that its occurrence is not linked to homosexual practices. These epidemiological features have been confirmed by our findings that HHV-8 is present in skin lesions from women and heterosexual men, ruling out the possibility that male homosexual activity is the exclusive mode of transmission of the virus.
Our detection of HHV-8 DNA in the semen obtained from heterosexual HIV-positive and HIV-negative males, including one with KS, is indicative of a sexual route of transmission for this virus. Owing to the small size of samples and bias of selection, it is not possible at present to make any comparison of the frequency of the positivities among the different groups and to draw any conclusion on the role of associated risk factors (number of sex partners, past history of other STD) which might influence the spread of the virus in the general population.
No HHV-8 DNA sequences occurred in cervical smears of women with normal or abnormal cytologies. This finding does not support the possibility of viral transmission through heterosexual contacts. The exiguity of the sample, a possible transient infection of the vaginal tract due to the low viral load of HHV-8 in semen of HIV-negative heterosexual males, or a lack of genital organ site of active viral replication might explain the negative results. In any case, at present, experimental evidence is lacking for a possible role of HHV-8 as cofactor for the development of premalignant and malignant lesions of the female genital tract.
Our data agree with those previously reported by Lin et al. [16] and Monini et al. [12], who have found HHV-8, with different frequencies, in ejaculates of HIV-positive and HIV-negative individuals. All these results focused attention on the low copies of virus detected in the specimens. The semen is probably not the site of an active viral replication but contains only a few cells carrying the virus. Moreover the virus may be shed intermittently, as recently suggested by Gupta et al. [19], who could not always detect HHV-8 in serial semen samples of HIV-positive homosexuals with KS.
Other researchers [17,18] have not found HHV-8 sequences in semen of HIV-positive and HIV-negative individuals. The discrepancy between their findings and our data cannot only be ascribed to the higher sensitivity of our PCR, which can detect at least five copies of HHV-8 DNA, because Corbellino et al. [18] used a very sensitive nested-PCR method. On the contrary, it is possible to propose a different circulation of HHV-8 in different geographical areas. Several papers show conflicting data on this topic, in part depending on whether antibodies to latent or lytic-cycle HHV-8 antigens were used [20-25].
At present, however, some conclusions can be drawn: first, the virus is predominantly sexually transmitted; second, differences in incidence of classic and endemic KS between different countries parallel the relative prevalence of the virus in their general population; and third, HHV-8 infection in individuals with HIV-induced immunosuppression confers to these patients a very high oncogenic risk [26]. Only well-designed sero-epidemiological surveys, carried out in several geographical areas, will be able to establish the exact prevalence of HHV-8 in general population.
HHV-8 was also detected in PBMC of HIV-positive and HIV-negative individuals. It must be stressed that the virus is present in the same individual not only in the semen but also in the circulating PBMC. Bigoni et al. [14] have established that PBMC of healthy individuals can carry HHV-8 sequences suggesting that the rare cells in the peripheral blood containing a low load of HHV-8 sequences correspond to latently or chronically infected cells. The immunosuppression may be the occasion of virus reactivation, since the viral load of HHV-8 in PBMC increased by 10- to 1000-fold in HIV-seropositive patients [14]. Our results agree with this hypothesis, since HHV-8 sequences could be detected easily by one-step PCR in PBMC of AIDS patients with and without KS, but only by two-step PCR in healthy individuals. HHV-8 detection in PBMC, however, may have a clinical relevance as predictor for the development of KS [27,28].
Since several reports [3,4] and our data have confirmed that HHV-8 sequences can be detected easily in classic and AIDS-associated KS, the problem is to ascertain whether HHV-8 is capable of causing KS by itself, or if the presence of HIV is required, and what role immunosuppression and various cytokines may play in the process. The exact relationship between the involvement of the immune system and the presence of HIV and/or HHV-8 in the development of a KS lesion is uncertain at this time [29].
Our findings confirm that coincident HIV infection is not always necessary for the development of KS and open the way to further investigations on environmental conditions, other than HIV, which might contribute to the clinical presentation of KS.
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